A better understanding of Mycobacterium tuberculosis pathogenesis requires improved understanding of the interactions between mycobacteria and their host cells. Clues to these interactions may be obtained by identifying the macrophage genes whose expression is induced or repressed following phagocytosis of M. tuberculosis. Additional clues to these interactions may come from genetic manipulation of the M. tuberculosis genome, leading to the identification of mycobacterial genes that play a role in disease pathogenesis. The goals of this proposal are i) to identify all macrophage genes whose expression levels are influenced by M. tuberculosis and 2) to develop vectors and techniques that permit genetic manipulation of the M. tuberculosis genome. To accomplish the first goal, the specific aims of this proposal are 1) to identify macrophage genes whose expression is induced or repressed following phagocytosis of M. bovis BCG and M. tuberculosis using a J774 macrophage model system, and 2) to determine whether the same genes in J774 macrophage and human alveolar macrophage are sensitive to BCG and M. tuberculosis. Differential screening of recombinant macrophage cDNA has already produced genes whose expression is influenced by phagocytosis of BCG or M. tuberculosis. To accomplish the second goal, the specific aims are 1) to further develop vectors and techniques that permit genetic manipulation of the BCG and M. tuberculosis genomes through homologous recombination, and 2) to use homologous recombination techniques to disrupt specific genes in the M. tuberculosis genome that may contribute to pathogenesis. Initial results indicate that homologous recombination can be accomplished in the slow- growing mycobacteria; improvements in these techniques should permit manipulation of the M. tuberculosis genome.